Cross-presentation of exogenous antigens by host professional antigen-presenting cells (APCs) plays a pivotal role in the initiation and development of T cell immune responses to tumor associated antigens, including self or mutated self-antigens derived from tumor cells, and foreign antigens derived from infectious agents. Prospective cancer vaccines have been developed that attempt to harness the cross-presentation of exogenous antigens to illicit a specific immune response against a tumor.
Autophagy is a cellular process in which portions of the cytoplasm are sequestered by double membrane vesicles termed autophagosomes that range in size from 0.5-2 μm. The contents of these autophagosomes are degraded in a lytic compartment, which facilitates the turnover of long-lived proteins and is critical for maintaining the pool of amino acids needed for anabolism. A key marker of the induction of autophagy is the conversion of the cytosolic form of microtubule-associated protein 1 light chain 3 (LC3-I) via a series of ubiquitin-like conjugation steps to the lipidated form (LC3-PE) that is tightly associated with autophagosomes. A recently described protein, p62/SQSTM1 (sequestosome or p62), binds both polyubiquitin and LC3 and thus facilitates degradation of ubiquitinated proteins via autophagy. Interaction of LC3 with p62 has added a layer of complexity to the autophagic network and suggests that this bulk degradation process may be more selective than previously appreciated.
Autophagy in tumor cells is important for efficient cross-presentation and subsequent induction of tumor immunity in a B16 melanoma model. Cross-presentation is significantly inhibited when autophagy is blocked, and increased when autophagy is promoted. Additionally, autophagosome-containing vesicles, termed DRibbles (DRiPs in blebs) isolated from tumors served as a potent antigen source in cross-presentation assays and in vivo vaccine studies. An important part of the process of generating the autophagosome-containing (DRibble) vaccine is the treatment of cells with bortezomib, which blocks the proteasome and results in an accumulation of ubiquitinated (Ub) proteins.
However, the inventors herein have recognized that the methods for production and isolation of DRibbles disclosed in the prior art may be insufficient to produce and isolate an enriched population of autophagosomes and their component material to be further utilized as an effective vaccine. Data points to ubiquitinated proteins, such as SLiPs and DRiPs, coupled with a chaperone (p62) as being responsible for the vaccine's potency. By isolating the ubiquitinated proteins, the highly-immunogenic portion of the autophagosome may be concentrated and effectively packaged as an immunogenic compound.
In one example, a method of inducing a specific immune response in a mammal, comprising: providing a first composition comprising isolated ubiquitinylated proteins in solution in the absence of membrane bound organelles, the isolated ubiquitinylated proteins comprising one or more specific antigens, and further comprising a threshold quantity of polyubiquitinylated short-lived proteins and polyubiquitinylated defective ribosomal products. The isolated ubiquitinylated proteins are affinity-purified from tumor-derived cells grown in culture, the tumor-derived cells being inhibited from degrading ubiquitinylated proteins via the proteasome while being grown in culture. In this way, highly immunogenic short-lived proteins and defective ribosomal products may be loaded onto dendritic cells for cross-presentation and priming of antigen-specific T cells restricted by either classical or non-classical MHC.
The above advantages and other advantages, and features of the present description will be readily apparent from the following Detailed Description when taken alone or in connection with the accompanying drawings.
It should be understood that the summary above is provided to introduce in simplified form a selection of concepts that are further described in the detailed description. It is not meant to identify key or essential features of the claimed subject matter, the scope of which is defined uniquely by the claims that follow the detailed description. Furthermore, the claimed subject matter is not limited to implementations that solve any disadvantages noted above or in any part of this disclosure.